title: Simultaneous ultrastructural analysis of fluorochrome-photoconverted diaminobenzidine and gold immunolabelling in cultured cells.
abstract: Diaminobenzidine photoconversion is a technique by which a fluorescent dye is transformed into a stably insoluble, brown, electrondense signal, thus enabling examination at both bright field light microscopy and transmission electron microscopy. In this work, a procedure is proposed for combining photoconversion and immunoelectron microscopy: in vitro cell cultures have been first submitted to photoconversion to analyse the intracellular fate of either fluorescent nanoparticles or photosensitizing molecules, then processed for transmission electron microscopy; different fixative solutions and embedding media have been used, and the ultrathin sections were finally submitted to post-embedding immunogold cytochemistry. Under all conditions the photoconversion reaction product and the target antigen were properly detected in the same section; Epon-embedded, osmicated samples required a pre-treatment with sodium metaperiodate to unmask the antigenic sites. This simple and reliable procedure exploits a single sample to simultaneously localise the photoconversion product and a variety of antigens allowing a specific identification of subcellular organelles at the ultrastructural level.
authors.affiliation: Dipartimento di Scienze Neurologiche e del Movimento, sezione di Anatomia e Istologia, Università di Verona. manuela.malatesta@univr.it.
authors.forename: M
authors.initials: M
authors.lastname: Malatesta
authors.forename: C
authors.initials: C
authors.lastname: Zancanaro
authors.forename: M
authors.initials: M
authors.lastname: Costanzo
authors.forename: B
authors.initials: B
authors.lastname: Cisterna
authors.forename: C
authors.initials: C
authors.lastname: Pellicciari
mesh_terms: D015100:3,3'-Diaminobenzidine / Q000737:chemistry; D000818:Animals; D002460:Cell Line; D002477:Cells / Q000648:ultrastructure*; D002478:Cells, Cultured; D005456:Fluorescent Dyes / Q000737:chemistry*; D006046:Gold / Q000737:chemistry*; D006367:HeLa Cells; D006801:Humans; D007150:Immunohistochemistry; D046529:Microscopy, Electron, Transmission; D051381:Rats
chemical_list: D005456:Fluorescent Dyes; D015100:3,3'-Diaminobenzidine; D006046:Gold